Abstract-Three well-defined laminar flow profiles were created to distinguish the influence of a gradient in shear and steady shear on platelet-derived growth factor A (PDGF-A) and monocyte chemoattractant protein-1 (MCP-1) expression in human endothelial cells. The flow profiles (16 dyne/cm 2 maximum shear stress) were ramp flow (shear stress smoothly transited at flow onset), step flow (shear stress abruptly applied at flow onset), and impulse flow (shear stress abruptly applied for 3 s only). Ramp flow induced only minor expression of PDGF-A and did not increase MCP-1 expression.Step flow increased PDGF-A and MCP-1 mRNA levels 3-and 2-fold at 1.5 hours, respectively, relative to ramp flow. In contrast, impulse flow increased PDGF-A and MCP-1 expression 6-and 7-fold at 1.5 hours, and these high levels were sustained for at least 4 hours. These results indicate that a temporal gradient in shear (impulse flow and the onset of step flow) and steady shear (ramp flow and the steady component of step flow) stimulates and diminishes the expression of PDGF-A and MCP-1, respectively. NO synthase inhibitor N G -amino-L-arginine (L-NAA) was found to markedly enhance MCP-1 and PDGF-A expression induced by step flow, but decrease their expression induced by impulse flow, in a dose-dependent manner. NO donor spermine-NONOate (SPR/NO) dose-dependently reduced the MCP-1 and PDGF-A expression induced by impulse flow. Moreover, impulse flow was found to stimulate sustained (4 hours) IB-␣ degradation and egr-1 mRNA induction. L-NAA prevented IB-␣ degradation, whereas SPR/NO increased IB-␣ resynthesis 2 hours after impulse flow. The exact nature and influence of local shear involved in endothelial dysfunction leading to susceptibility for atherogenesis, however, remains unclear. Endothelial cells (EC) throughout the vasculature experience a variety of flow environments both with spatial variances and with temporal gradients in wall shear stress. In the venous system wall shear stress is lower and minimal gradients in shear stress exist because of the nonpulsatile nature of the blood flow. In the arterial system the flow conditions are generally assumed to be laminar and to present the endothelium with a high mean wall shear stress in addition to large temporal gradients in shear stress. At arterial bifurcations and curvatures, locations known to be highly prone to atherogenesis, disturbed flow patterns may develop that result in low mean wall shear stress, but still present the EC with large temporal gradients in shear stress. 3 These observations, combined with other in vitro evidence, 4 -6 suggest that gradients in shear and steady shear represent different biomechanical stimuli that differentially regulate local endothelial function by distinct signaling pathway, and thus contribute to the characteristic distribution pattern of atherosclerosis.A host of endothelial genes exhibit differential responses to shear stress stimuli that may be involved in the focal localization of atherogenic plaques. 7 The application of step shear...

Background —The effect of temporal and spatial gradients in shear on primary human endothelial cell (HUVEC) proliferation was investigated. The sudden-expansion flow chamber (SEFC) model was used to differentiate the effect of temporal gradients in shear from that of spatial gradients. With a sudden onset of flow, cells are exposed to both temporal and spatial gradients of shear. The temporal gradients can be eliminated by slowly ramping up the flow. Methods and Results —HUVEC proliferation in the SEFC remained unstimulated when the onset of flow was slowly ramped. Sudden onset of flow stimulated a 105% increase of HUVEC proliferation (relative to ramped onset) within the region of flow reattachment. To further separate temporal and spatial gradients, a conventional parallel-plate flow chamber was used. A single 0.5-second impulse of 10 dyne/cm 2 increased HUVEC proliferation 54±3% relative to control. When flow was slowly ramped over 30 seconds, HUVEC proliferation was not significantly different from controls. Steady laminar shear over 20 minutes inhibited HUVEC proliferation relative to controls regardless of step (36±8%) or ramp (21±5%) onsets of flow. Conclusions —The results indicate that temporal gradients in shear stress stimulate endothelial cell proliferation, whereas spatial gradients affect endothelial proliferation no differently than steady uniform shear stress.

GTP cyclohydrolase 1 (GCH1) is rate limiting in the provision of the cofactor tetrahydrobiopterin for biosynthesis of catecholamines and NO. We asked whether common genetic variation at GCH1 alters transmitter synthesis and predisposes to disease. Here we undertook a systematic search for polymorphisms in GCH1, then tested variants' contributions to NO and catecholamine release as well as autonomic function in twin pairs. Renal NO and neopterin excretions were significantly heritable, as were baroreceptor coupling (heart rate response to BP fluctuation) and pulse interval (1/heart rate). Common GCH1 variant C+243T in the 3'-untranslated region (3'-UTRs) predicted NO excretion, as well as autonomic traits: baroreceptor coupling, maximum pulse interval, and pulse interval variability, though not catecholamine secretion. In individuals with the most extreme BP values in the population, C+243T affected both diastolic and systolic BP, principally in females. In functional studies, C+243T decreased reporter expression in transfected 3'-UTRs plasmids. We conclude that human NO secretion traits are heritable, displaying joint genetic determination with autonomic activity by functional polymorphism at GCH1. Our results document novel pathophysiological links between a key biosynthetic locus and NO metabolism and suggest new strategies for approaching the mechanism, diagnosis, and treatment of risk predictors for cardiovascular diseases such as hypertension.